exRNA in Colorectal Carcinoma: Biogenesis and Function Project 2. Mechanisms and Functional Consequences of Selective miRNA transfer via extracellular vesicles James G. Patton - Investigator Summary Increasing evidence supports the hypothesis that extracellular vesicles (EVs) constitute a novel form of cell-cell communication through the transfer of protein, RNA and lipid cargo. Project 2 will focus on selective EV miRNA transport and uptake by recipient cells. We previously showed that EVs from KRAS mutant cells are enriched in miR-100 and miR-125b and we have now shown in 3D culture that EVs enriched in miR-100 and miR-125b can confer cetuximab resistance in recipient cells. However, it remains unknown how miR-100 and miR-125b (as well as other miRNAs) are selectively targeted for secretion while other miRNAs are retained in cells. Here, we will focus on determining whether transfer of miR-100 and miR-125b can alter the tumor microenvironment using a novel in vivo xenograft model in zebrafish. We will also use an adaptation of CRISPR-Display to test the hypothesis that specific RNA sequences and/or base modifications regulate selective miRNA export. The same cell culture assay will be used to identify RNA binding proteins (in concert with Project 1) that recognize sequence motifs or modified bases to drive secretion of specific miRNAs which will then be extended to in vivo effects using the zebrafish xenograft model. Lastly, our current hypothesis is that transfer of miR-100 and miR-125b results in the activation of Wnt signaling but the full range of mRNA targets for these miRNAs remains unknown. Thus, we will use RIP-USE to combine immunoprecipitation of Ago2 associated miRNAs with differential expression analysis using Unbiased Sequence Enrichment (RIP-USE) to identify all targets of miR-100 and miR-125b. Normal cell-cell interactions and stem cell niches in the colonic crypt appear to result from the secretion of EVs that set up opposing gradients of Wnt and EGFR signaling, our analyses will identify potential therapeutic target genes whose expression is altered when proper cell-cell communication is altered during colorectal cancer.